Genomic DNA Samples Research Articles

Ultrasensitive isothermal detection of a plant pathogen by using a gold nanoparticle-enhanced microcantilever sensor

• GNP-enhanced dynamic microcantilever biosensor is constructed for pathogen analysis. • Combined with RPA, a LOD as low as one copy genomic DNA could be achieved. • This RPA-MCL could detect 57 ppm pathogen genomic DNA out of oilseed plant. Sensitive and rapid detection of plant pathogens, particularly for on-site diagnosis during custom inspections, is essential to prevent and control the spread of these pathogens. To address the need, a combined assay for detecting plant pathogens was developed by using gold nanoparticle-enhanced dynamic microcantilever (MCL) biosensing and isothermal recombinase polymerase amplification (RPA). In this study, we used Leptosphaeria maculans (L . maculans) , an aggressive fungus responsible for major losses in oilseed rape yield worldwide, as the target pathogen to demonstrate the merits of the combined RPA and MCL strategy (named as the RPA-MCL assay). The results indicated that the sensitivity of the RPA-MCL assay is higher than that of the fluorescence RPA assay, with the detection limit of only one copy of L . maculans DNA. In the practical assay, the newly developed RPA-MCL method was found to detect 57 ppm L . maculans genomic DNA in the oilseed rape seeds genomic DNA sample. Considering the high sensitivity and specificity of this strategy, we envision that the proposed RPA-MCL assay could have wide applications in nucleic acid diagnostics for on-site plant pathogen detection and species identification.

Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus

The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr-8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing “resistant” allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous “resistant” genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the “resistant” allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.

Optimization of diagnostic strategy for non-invasive cell-free foetal RHD determination from maternal plasma.

The aim of the study was to optimize routine non-invasiveprenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetalRHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.

Seroprevalence and Genotypic Analysis of Ehrlichia canis Infection in Dogs and Humans in Cauca, Colombia.

Ehrlichia canis infections have been reported in humans in Venezuela and Costa Rica. In this study, 506 healthy residents and 114 dogs from four municipalities (Cauca, Colombia) were surveyed and blood samples collected. Antibodies to E. canis in human and canine sera were evaluated using the Tandem repeat protein 19 (TRP19) peptide ELISA and indirect immunofluorescence assay (IFA). Ehrlichia canis TRP19 antibodies were detected in only 1/506 human sera, but the single positive sample was negative by IFA. The majority (75/114; 66%) of dogs surveyed had antibodies to the E. canis TRP19 peptide by ELISA, and eight randomly selected sera were further confirmed by E. canis IFA. Genomic DNA samples obtained from 73 E. canis TRP19 ELISA-positive dog blood samples were examined by PCR targeting the 16S ribosomal ribonucleic acid (rRNA) gene. Ehrlichia canis 16S rRNA was amplified in 30 (41%) of the dogs, and 16 amplicons were selected for DNA sequencing, which confirmed that all were E. canis. A second PCR was performed on the 16 confirmed E. canis 16S rRNA PCR-positive samples to determine the TRP36 genotype by amplifying the trp36 gene. TRP36 PCR amplicon sequencing identified nine dogs infected with the U.S. E. canis TRP36 genotype (56%), one dog with the Brazilian genotype (6%), and six dogs with the Costa Rican genotype (38%). Moreover, these molecular genotype signatures were consistent with serologic analysis using TRP36 genotype-specific peptides. Notably, there was no serologic evidence of E. canis infection in humans, suggesting that E. canis infection in dogs in Cauca is not associated with zoonotic human infection.

Metagenomic Snapshots of Viral Components in Guinean Bats.

To prevent the emergence of zoonotic infectious diseases and reduce their epidemic potential, we need to understand their origins in nature. Bats in the order Chiroptera are widely distributed worldwide and are natural reservoirs of prominent zoonotic viruses, including Nipah virus, Marburg virus, and possibly SARS-CoV-2. In this study, we applied unbiased metagenomic and metatranscriptomic approaches to decipher the virosphere of frugivorous and insectivorous bat species captured in Guéckédou, Guinea, the epicenter of the West African Ebola virus disease epidemic in 2013–2016. Our study provides a snapshot of the viral diversity present in these bat species, with several novel viruses reported for the first time in bats, as well as some bat viruses closely related to known human or animal pathogens. In addition, analysis of Mops condylurus genomic DNA samples revealed the presence of an Ebola virus nucleoprotein (NP)-derived pseudogene inserted in its genome. These findings provide insight into the evolutionary traits of several virus families in bats and add evidence that nonretroviral integrated RNA viruses (NIRVs) derived from filoviruses may be common in bat genomes.

Improving the Understanding of the Immunopathogenesis of Lymphopenia as a Correlate of SARS-CoV-2 Infection Risk and Disease Progression in African Patients: Protocol for a Cross-sectional Study.

BackgroundThe COVID-19 pandemic, caused by SARS-CoV-2, continues to impact health systems throughout the world with serious medical challenges being imposed on many African countries like Nigeria. Although emerging studies have identified lymphopenia as a driver of cytokine storm, disease progression, and poor outcomes in infected patients, its immunopathogenesis, as well as environmental and genetic determinants, remain unclear. Understanding the interplay of these determinants in the context of lymphopenia and COVID-19 complications in patients in Africa may help with risk stratification and appropriate deployment of targeted treatment regimens with repurposed drugs to improve prognosis.ObjectiveThis study is designed to investigate the role of vitamin D status, vasculopathy, apoptotic pathways, and vitamin D receptor (VDR) gene polymorphisms in the immunopathogenesis of lymphopenia among African people infected with SARS-CoV-2.MethodsThis cross-sectional study will enroll 230 participants, categorized as “SARS-CoV-2 negative” (n=69), “COVID-19 mild” (n=32), “hospitalized” (n=92), and “recovered” (n=37), from two health facilities in Lagos, Nigeria. Sociodemographic data, travel history, and information on comorbidities will be obtained from case files and through a pretested, interview-based structured questionnaire. Venous blood samples (5 mL) collected between 8 AM and 10 AM and aliquoted into EDTA (ethylenediaminetetraacetic acid) and plain tubes will be used for complete blood count and CD4 T cell assays to determine lymphopenia (lymphocyte count <1000 cells/µL) and CD4 T lymphocyte levels, as well as to measure the concentrations of vitamin D, caspase 3, soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble Fas ligand (sFasL) using an autoanalyzer, flow cytometry, and ELISA (enzyme-linked immunosorbent assay) techniques. Genomic DNA will be extracted from the buffy coat and used as a template for the amplification of apoptosis-related genes (Bax, Bcl-2, BCL2L12) by polymerase chain reaction (PCR) and genotyping of VDR (Apa1, Fok1, and Bsm1) gene polymorphisms by the PCR restriction fragment length polymorphism method and capillary sequencing. Total RNA will also be extracted, reverse transcribed, and subsequently quantitated by reverse transcription PCR (RT-PCR) to monitor the expression of apoptosis genes in the four participant categories. Data analyses, which include a test of association between VDR gene polymorphisms and study outcomes (lymphopenia and hypovitaminosis D prevalence, mild/moderate and severe infections) will be performed using the R statistical software. Hardy-Weinberg equilibrium and linkage disequilibrium analyses for the alleles, genotypes, and haplotypes of the genotyped VDR gene will also be carried out.ResultsA total of 45 participants comprising 37 SARS-CoV-2–negative and 8 COVID-19–recovered individuals have been enrolled so far. Their complete blood counts and CD4 T lymphocyte counts have been determined, and their serum samples and genomic DNA and RNA samples have been extracted and stored at –20 °C until further analyses. Other expected outcomes include the prevalence and distribution of lymphopenia and hypovitaminosis D in the control (SARS-CoV-2 negative), confirmed, hospitalized, and recovered SARS-CoV-2–positive participants; association of lymphopenia with CD4 T lymphocyte level, serum vitamin D, sVCAM-1, sFasL, and caspase 3 levels in hospitalized patients with COVID-19; expression levels of apoptosis-related genes among hospitalized participants with COVID-19, and those with lymphopenia compared to those without lymphopenia; and frequency distribution of the alleles, genotypes, and haplotypes of VDR gene polymorphisms in COVID-19–infected participants.ConclusionsThis study will aid in the genotypic and phenotypic stratification of COVID-19–infected patients in Nigeria with and without lymphopenia to enable biomarker discovery and pave the way for the appropriate and timely deployment of patient-centered treatments to improve prognosis.International Registered Report Identifier (IRRID)DERR1-10.2196/21242

The complete mitochondrial genome of Sanghuangporus vaninii Zhehuang-1 (Hymenochaetales, Basidiomycota)

In this study, the complete mitochondrial genome of Sanghuangporus vaninii Zhehuang-1 was determined. Genomic DNA samples individually collected from a population in southeastern China (Qiandaohu, Zhejiang Province, N29°44′24′′, E118°52′48′′) were sequenced with an Illumina NovaSeq 6000. The complete mitochondrial genome was 97,345 bp in length and consisted of 22 protein-coding genes (PCGs), 29 tRNAs, 3 ribosomal RNAs, and a control region. Sanghuangporus vaninii has a mitochondrial gene arrangement that is similar to that of S. sanghuang. Phylogenetic analysis performed using ML methods based on the complete mitogenome sequence showed that S. vaninii is a member of the Polyporaceae. The complete mitogenome sequence provides important data for further study of the Inonotus linteus complex.

RNF213 rare variants and cerebral arteriovenous malformation in a Chinese population

ObjectiveCerebral arteriovenous malformation (AVM) is characterised by an abnormal tangle of arteries and veins, the rupture of which is a significant portion of the morbidity and mortality cases, especially in young populations. However, the exact risk factors and pathophysiologic mechanisms of AVM remain poorly understood. RNF213 variants have been identified as obvious susceptible factors of several cerebrovascular disorders, such as Moyamoya disease and intracranial aneurysms. Thus, this study aimed to determine whether there is an association between RNF213 rare variants and AVM. MethodsThe AVM group included 22 patients with AVM. The control group included 1007 samples from the GeneSky in-house database and 208 samples from the 1000 Genome Project of Chinese Han Population. Genomic DNA samples were extracted from the peripheral blood of the AVM patients, and targeted exome sequencing of RNF213 was performed to assess the existence of low-frequency or rare variants. Sanger sequencing was performed to validate the identified variants. Logistic regression analysis was performed to calculate the odds ratios (ORs) and 95 % confidence intervals (CIs) of the candidate variants and risk of AVM. Statistical analyses were performed using SPSS version 21.0. ResultsThe RNF213 c.10997T>C variant (amino acid mutation p.M3666T, NM_001256071) was observed in two AVM patients after filtration. It was significantly associated with AVM in the Chinese population (ORs, 10.30 and 25.08; 95 %; CIs, 1.38–77.10 and 4.34–144.90 compared with 1000 Genome Project of Chinese Han Population and GeneSky in-house database, respectively). ConclusionRare variants of RNF213 are associated with AVM in the Chinese population, suggesting the important role of RNF213 in AVM. Further studies are needed to verify these findings.

No impact of soluble epoxide hydrolase rs4149243, rs2234914 and rs751142 genetic variants on the development of type II diabetes and its hypertensive complication among Jordanian patients.

Human soluble epoxide hydrolase plays a major role in cardiovascular homoeostasis. Genetic variants in the EPHX2 gene among different ethnic groups are associated with cardiovascular complications, such as hypertension. However, no reports regarding the association of EPHX2 genotype with hypertension among type II diabetic (T2D) patients of Middle Eastern Jordanian origin exist. The current study aimed to elucidate the association of the EPHX2 allele, genotype and haplotype with T2D, hypertension and parameters of lipid profile parameters among Jordanian T2D patients. Ninety-three genomic DNA samples of non-diabetic controls and 97 samples from T2D patients were genotyped for EPHX2 rs4149243, rs2234914 and rs751142 genetic variants. The DNA samples were amplified using polymerase chain reaction (PCR) and then sequenced using Applied Biosystems Model (ABI3730x1). The functionality of intronic EPHX2 variants was predicted using the in silico Berkely Drosophila Genome Project software. We found no significant (P>.05) association between the EPHX2 rs4149243, rs2234914 and rs751142 allele, genotype and haplotype and the incidence of T2D and hypertension. Additionally, no association (P>.05) between these EPHX2 genetic variants with the baseline total cholesterol, low- and high-density lipoproteins and triglycerides among both non-diabetic and diabetic volunteers was found. However, we found an inter-ethnic variation (χ2 -test, P value˂.05) in the allele frequency of the EPHX2 rs4149243 and rs2234914 variants between Jordanians and other ethnic populations. Also, the in silico Berkely Drosophila Genome Project software predicted that the intronic EPHX2 rs4149243 could alter the splicing of intron 7. It can be concluded from this study that EPHX2 rs4149243, rs2234914 and rs751142 genetic variants do not play a role in the development of T2D and hypertension among Jordanian T2D patients. Further genetic studies with larger sample sizes are needed to find out the association of other functional EPHX2 variants with cardiovascular diseases among T2D patients in Jordan.

A Rapid PCR-Free Next-Generation Sequencing Method for the Detection of Copy Number Variations in Prenatal Samples.

Next-generation sequencing (NGS) is emerging as a new method for the detection of clinically significant copy number variants (CNVs). In this study, we developed and validated rapid CNV-sequencing (rCNV-seq) for clinical application in prenatal diagnosis. Low-pass whole-genome sequencing was performed on PCR libraries prepared from amniocyte genomic DNA. From 10–40 ng of input DNA, PCR-free libraries consistently produced sequencing data with high unique read mapping ratios, low read redundancy, low coefficient of variation for all chromosomes and high genomic coverage. In validation studies, reliable and accurate CNV detection using PCR-free-based rCNV-seq was demonstrated for a range of common trisomies and sex chromosome aneuploidies as well as microdeletion and duplication syndromes. In reproducibility studies, CNV copy number and genomic intervals closely matched those defined by chromosome microarray analysis. Clinical testing of genomic DNA samples from 217 women referred for prenatal diagnosis identified eight samples (3.7%) with known chromosome disorders. We conclude that PCR-free-based rCNV-seq is a sensitive, specific, reproducible and efficient method that can be used in any NGS-based diagnostic laboratory for detection of clinically significant CNVs.

Novel variants in TBC1D24 associated with epilepsy and deafness: Report of two cases

To identify the causative variants in two unrelated Chinese patients presenting with epilepsy and deafness. The two patients underwent a thorough examination, including brain MRI, EEG and metabolic studies. Next-generation sequencing (NGS) was performed on genomic DNA samples from the siblings and parents. Sanger sequencing was used to confirm the variants. Gene sequencing revealed that they carried two novel compound heterozygous missense variants of the TBC1D24: c.116 C>T (p.Ala39Val) and c.827T>C (p.Ile276Thr) in patient 1; c.404 C>T (p.Pro135Leu) and c.679T>C (p.Arg227Trp) in patient 2. Audiologic examination showed bilateral sensorineural hearing loss in both patients. We have found novel variants in the TBC1D24 in two Chinese unrelated patients. They result in a rare phenotype, characterized by drug-resistant epilepsy and deafness.

Molecular Fingerprinting of Indian Medicinal Tree Sara Asoca using RAPD Markers

Saraca asoca is an important medicinal tree facing a serious problem of reduction from its instinctivetenancy in India.Before formulation of conservation strategies for geographical protection of S. asoca genotypes available in India, it is necessary to characterize them. In the current study, the RAPD markers have been utilized effectively for categorization of S. asoca collected from 15 diverse sites of India. An initial experiment on the amplification suitability of genomic DNA samples of four S. asoca was done with 35RAPD primers. Among them only twenty sixproved their efficiency in two times repeat amplification.Total 146 bands were amplified and out of these 97 bands were found to be polymorphic. The average numbers of total band was 5.61 while average numbers of polymorphic bands was 3.73. The numbers of bands produced per primer ranged from 3 (OPE-15) to 8 (RUF205). Among all studied markers the highest percentage (100%) of polymorphism was demonstrated by only one marker (OPE-06). The lowest percent of polymorphicm (20%) was demonstrated by marker RUF211. The average percentage of polymorphism was 66.44%. Cluster analysis grouped all the S. asoca genotypes under study into two groups. Grouping of genotypes according to their sites of collection demonstrates higher similarity among or between them. The results obtained in the current study may help to formulate conservation strategies for the conservation of S. asoca genotypes.

Composition and Potential Function of Fecal Bacterial Microbiota from Six Bird Species

Gut microbial communities play a fundamental role in health and disease, but little is known about the gut microbiota of pet bird species. This is important to better understand the impact of microbes on birds’ health but may also be relevant in a context of zoonoses. Total genomic DNA samples from pooled fecal samples from 30 flocks (4–7 pet birds per flock) representing over 150 birds of six different species (two Passeriformes: Northern Mockingbird (Mimus polyglottos) and Zebra Finch (Taeniopygia guttata), and four Psittaciformes: Lovebird (Agapornis, different species), Cockatiel (Nymphicus hollandicus), Red-rumped Parrot (Psephotus haematonotus), and Rose-ringed Parakeet (Psittacula krameri) were used for 16S rRNA gene analysis. Several taxa were found to be different among the bird species (e.g., lowest median of Lactobacillus: 2.2% in Cockatiels; highest median of Lactobacillus: 79.4% in Lovebirds). Despite marked differences among individual pooled samples, each bird species harbored a unique fecal bacterial composition, based on the analysis of UniFrac distances. A predictive approach of metagenomic function and organism-level microbiome phenotypes revealed several differences among the bird species (e.g., a higher proportion of proteobacteria with the potential to form biofilms in samples from Northern Mockingbirds). The results provide a useful catalog of fecal microbes from pet birds and encourage more research on this unexplored topic.

Clinical relevance of oncogenic driver mutations identified in endometrial carcinoma

PurposeEndometrial carcinoma (EC) is a clinically heterogeneous disease characterized by a number of different histological subtypes, and its heterogeneity may be involved in the accumulation of multiple genetic alterations. The aim of this work was to investigate the comprehensive mutational profile of EC tumors, and examine the associations between somatic mutations and clinicopathological features or survival in EC patients. MethodsA total of 100 surgical tumors were obtained from EC patients who had previously undergone surgery. Genomic DNA samples extracted from fresh-frozen tissues were analyzed using the Ion AmpliSeq Cancer Hotspot Panel v2 Kit, covering 50 tumor-related genes. ResultsValidated mutations were detected in 91 of the 100 tumors (91%) and identified in eight of the most frequently mutated genes, namely PTEN (57%), PIK3CA (51%), TP53 (30%), KRAS (23%), CTNNB1 (21%), FBFR2 (13%), FBXW7(10%) and RB1 (9%). PTEN mutations were found to associated with young age (< 60), early-stage, endometrioid histology, non-recurrence and better overall survival (OS). CTNNB1 mutations were associated with young age, endometrioid histology and better OS. On the other hands, TP53 mutations were associated with late-stage, non-endometrioid histology, high-grade, recurrence and worse OS. FBWX7 mutations were associated with late-stage, vascular invasion and lymph node metastasis. FGFR2 mutations correlated with deep (≥ 1/2) myometrial invasion. ConclusionOur comprehensive mutational profile will be useful for understanding and evaluating the molecular characteristics of EC tumors, and may lead to the establishment of novel treatment strategies that improve the survival of patients with EC in the future.

Fine-Scale Genetic Structure and Demographic History in the Miyako Islands of the Ryukyu Archipelago

The Ryukyu Archipelago is located in the southwest of the Japanese islands and is composed of dozens of islands, grouped into the Miyako Islands, Yaeyama Islands, and Okinawa Islands. Based on the results of principal component analysis on genome-wide single-nucleotide polymorphisms, genetic differentiation was observed among the island groups of the Ryukyu Archipelago. However, a detailed population structure analysis of the Ryukyu Archipelago has not yet been completed. We obtained genomic DNA samples from 1,240 individuals living in the Miyako Islands, and we genotyped 665,326 single-nucleotide polymorphisms to infer population history within the Miyako Islands, including Miyakojima, Irabu, and Ikema islands. The haplotype-based analysis showed that populations in the Miyako Islands were divided into three subpopulations located on Miyakojima northeast, Miyakojima southwest, and Irabu/Ikema. The results of haplotype sharing and the D statistics analyses showed that the Irabu/Ikema subpopulation received gene flows different from those of the Miyakojima subpopulations, which may be related with the historically attested immigration during the Gusuku period (900 − 500 BP). A coalescent-based demographic inference suggests that the Irabu/Ikema population firstly split away from the ancestral Ryukyu population about 41 generations ago, followed by a split of the Miyako southwest population from the ancestral Ryukyu population (about 16 generations ago), and the differentiation of the ancestral Ryukyu population into two populations (Miyako northeast and Okinawajima populations) about seven generations ago. Such genetic information is useful for explaining the population history of modern Miyako people and must be taken into account when performing disease association studies.

Effect of ACTN3 R577X Genotype on Injury Epidemiology in Elite Endurance Runners.

The p.R577X polymorphism (rs1815739) in the ACTN3 gene causes individuals with the ACTN3 XX genotype to be deficient in functional α-actinin-3. Previous investigations have found that XX athletes are more prone to suffer non-contact muscle injuries. This investigation aimed to determine the influence of the ACTN3 R577X polymorphism in the injury epidemiology of elite endurance athletes. Using a cross-sectional experiment, the epidemiology of running-related injuries was recorded for one season in a group of 89 Spanish elite endurance runners. ACTN3 R577X genotype was obtained for each athlete using genomic DNA samples. From the study sample, 42.7% of athletes had the RR genotype, 39.3% had the RX genotype, and 18.0% had the XX genotype. A total of 96 injuries were recorded in 57 athletes. Injury incidence was higher in RR runners (3.2 injuries/1000 h of running) than in RX (2.0 injuries/1000 h) and XX (2.2 injuries/1000 h; p = 0.030) runners. RR runners had a higher proportion of injuries located in the Achilles tendon, RX runners had a higher proportion of injuries located in the knee, and XX runners had a higher proportion of injuries located in the groin (p = 0.025). The ACTN3 genotype did not affect the mode of onset, the severity, or the type of injury. The ACTN3 genotype slightly affected the injury epidemiology of elite endurance athletes with a higher injury rate in RR athletes and differences in injury location. However, elite ACTN3 XX endurance runners were not more prone to muscle-type injuries.

The associations of hostility and defensiveness with telomere length are influenced by sex and health status

BackgroundShorter telomere length (TL) may indicate premature cellular aging and increased risk for disease. While there is substantial evidence for shorter TL in individuals suffering from psychiatric disorders, data is scarce on maladaptive personality traits related to coronary artery disease (CAD). The purpose of this study was to evaluate the association of TL with hostility and defensiveness in individuals with CAD or other non-cardiovascular illnesses and whether associations were moderated by CAD status and sex.MethodsOne thousand thirty-six individuals (Mage = 65.40 ± 6.73 years) with and without CAD completed the Marlowe-Crowne Social Desirability Scale and the Cook–Medley Hostility Scale. Relative TL was measured via quantitative polymerase chain reaction of total genomic DNA samples. Analyses involved hierarchical regressions on TL, performed separately for hostility and defensiveness, controlling for pertinent sociodemographic, behavioural, and medical risk factors. Separate analyses were performed on 25 healthy participants.ResultsA hostility by sex interaction emerged (β = − .08, p = .006) in the patient groups, where greater hostility was associated with shorter TL in women only (p < .01). A Defensiveness by CAD status interaction (β = − .06, p = .049) revealed longer TL in more defensive CAD patients only (p = .06). In healthy men, shorter TL was observed in those with greater defensiveness (β = .52, p = .006) but lower hostility (β = − .43, p = .049).ConclusionHostility and defensiveness are differentially associated with TL as a function of sex and health status. The implication of these results for health remains to be determined, but propose an additional pathway through which the effect of maladaptive personality traits may contribute to CV and other disease.

Peripheral Blood BRCA1 Methylation Positively Correlates with Major Alzheimer's Disease Risk Factors.

Recent biomarker studies demonstrated that the central nervous system (CNS) environment can be observed from peripherally-derived samples. In a previous study, we demonstrated significant hypomethylation of the BRCA1 promoter region in neuronal cells from post-mortem brains of Alzheimer's disease patients through neuron-specific methylome analysis. Thus, we investigate the methylation changes in the BRCA1 promoter region in the blood samples. To analyze the methylation level of the BRCA1 promoter in peripheral blood from AD patients and normal controls. Genomic DNA samples from peripheral blood were obtained from the J-ADNI repository, and their biomarker data were obtained J-ADNI from the National Bioscience Database Center. Genomic DNA samples from an independent cohort for validation was obtained from Niigata University Hospital (Niigata, Japan). Amyloid positivity was defied by visual inspection of amyloid PET or a CSF Aβ42 value ≤ 333 pg/mL at the baseline. Methylation level of the BRCA1 promoter was analyzed by pyrosequencing. Compared to normal controls, methylation of the BRCA1 promoter in AD patients was not significantly changed; however, in AD patients, it showed a positive correlation with AD risk factors. Our data confirmed the importance of cell-type specific methylome analysis and also suggested that environmental changes in the CNS can be detected by observing the peripheral blood, implying that the peripheral BRCA1 methylation level can be a surrogate for AD.

Molecular Mechanisms of Resistance to Osimertinib from Liquid Biopsies of Plasma and Pleura Effusion in EGFR Mutant NSCLC Patients

In the past decade, tyrosine kinase inhibitors (TKI) have been developed and widely-approved by countries to treat advanced lung cancer patients with sensitizing epidermal growth factor (EGFR) mutations, and substantially improved patients’ prognosis compared to traditional chemotherapies. However, a large proportion of patients who initially experienced ostensibly remarkable therapeutic outcomes still developed inevitable drug resistance eventually, including those received newly developed third-generation TKIs. Previous studies have demonstrated the value of plasma cell-free DNA (cfDNA) in monitoring patients’ response to therapies in lung cancer. However due to several physiological reasons and DNA degradation, DNA released from tumor cell apoptosis in lung was hard to maintain at a high concentration and good quality before being collected from venous blood, leading to the loss of pivotal mutational information. Pleural effusion is a potential substitute to overcome these problems. However, limited studies have been reported to characterize the consistency of genetic alterations found in these two sample types, and due to the modest sample sizes, the mechanisms underlying third-generation TKI resistance remains elusive. Herein we drew 8ml venous blood and collected 20ml pleural effusion by pleural effusion drainage from 8 lung cancer patients, and performed hybrid-capture based next generation sequencing assays on 18 matched plasma cfDNA, pleural effusion cells genomic DNA and pleural effusion supernatant cfDNA samples, we have shown that only sequencing either plasma or pleural effusion sample resulted in the missing of mutations in key cancer drivers. Sequencing both pleural effusion and plasma yielded a more concrete genetic landscape for monitoring tumor response. We have also exhibited that somatic mutations and copy number changes in PI3K/AKT pathway, NOTCH signaling pathway, hedgehog signaling pathway and pathways involved in DNA damage repair might be potential mechanisms underlying TKI resistance, and might serve as candidate targets for developing novel therapies to circumvent patients’ resistance.

Segmental Duplication as Potential Biomarkers for Non-Invasive Prenatal Testing of Aneuploidies

Background: Segmental duplication (SD) regions are distinct targets for aneuploidy detection owing to the virtual elimination of amplification bias. The difficulty of searching SD sequences for assay design has hampered their applications. Methods:We developed a computational program, ChAPDes, which integrates SD searching, refinement, and design of specific PCR primer/probe sets in a pipeline to remove most of the manual work. The generated primer/probe sets were first tested in a multiplex multicolor melting curve analysis for the detection of five common aneuploidies. The primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21. Finally, a digital PCR protocol was established to quantify maternal plasma DNA sequences for the non-invasive prenatal detection of fetal trisomy 21. Findings: ChAPDes could output 21,772 candidate primer/probe sets for trisomy 13, 18, 21 and sex chromosome aneuploidies within 2 working days. Clinical evaluation of the multiplex multicolour melting curve analysis involving 463 fetal genomic DNA samples revealed a sensitivity of 100% and specificity of 99.64% in comparison with the reference methods. Using the established digital PCR protocol, we correctly identified two trisomy 21 fetuses and thirteen euploid foetuses from the maternal plasma samples. Interpretation: The combination of ChAPDes with digital PCR detection could facilitate the use of SD as potential biomarkers for the non-invasive prenatal testing of fetal chromosomal aneuploidies. Funding Information: This work was supported by the National Key Research & Developmental Program of China (2018YFC1003601), the National Natural Science Foundation of China (8167210 to Q.H.), the Science and Technology Project of Xiamen City (3502Z20183013), and the Guangxi Key Laboratory Thalassemia Research Project (20-065-20). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The collection of specimens and associated clinical data used in this study were approved by the Affiliated Suzhou Hospital of Nanjing Medical University and Suzhou Municipal Hospital. All patients who participated in this study provided written informed consent prior to enrollment and specimen collection. This study was performed in accordance with the principles of the Helsinki Declaration.